7. Project Summary Ergot alkaloids improve human health as powerful and versatile pharmaceuticals for treatment of multiple conditions including senile dementia, Alzheimer's disease, Parkinson's disease, migraines, hyperprolactinemia, and type 2 diabetes. Tremendous diversity in structure and activity can be found among the natural and semi-synthetic ergot alkaloids. Small changes in structure may lead to a great changes in activity. The long-term goal of this research program is to understand and ultimately control the biosynthesis of diverse ergot alkaloids by determining the functions of genes and gene products that produce and diversify ergot alkaloids. One goal of this particular project is to understand and control the biosynthesis of lysergic acid amides. Many of the more important pharmaceutical ergot alkaloids are lysergic acid amides or could be derived from lysergic acid amides. An understanding of the genes involved in making this class of ergot alkaloids is now more accessible because of their recent discovery in an experimentally tractable fungus, Metarhizium anisopliae. A second goal of this project is to test whether M. anisopliae will secrete pharmaceutically important and novel ergot alkaloids similar to the way it secreted its natural lysergic acid amides. This point is particularly relevant to industrial applications of this research, because ergot alkoid-producing fungi typically retain their ergot alkaloids in the solid phase of the culture, complicating purification. Specific aims of the proposed project are to: 1) Characterize the biosynthesis and diversification of lysergic acid amides; and, 2) Investigate Metarhizium anisopliae as a platform for production and modification of lysergic acid derivatives. The experimental approach to Aim 1 will focus on two novel genes associated with lysergic acid amides. The roles of the two genes will be ascertained through heterologous expression and gene knockout studies. The experimental approach to Aim 2 will involve application of knowledge of the functions of several ergot alkaloid pathway genes to engineer M. anisopliae to produce and potentially secrete important ergot alkaloids lysergic acid and dihydrolysergic acid, as well as a completely novel lysergic acid amide. The fungal strains produced by these genetic manipulations will be analyzed by molecular and biochemical methods to determine how expression or alteration of the candidate genes has affected the fungus's ergot alkaloid profile. Results of the proposed project will reveal roles of specific genes and provide strains of M. anisopliae that produce molecules with pharmaceutical significance. Additional benefits include meaningful experiences for graduate and undergraduate students and further development of a platform for modification and improvement of additional or novel ergot alkaloids.